PLZF-mediated control on VLA-4 expression in normal and leukemic myeloid cells.

The promyelocytic leukemia zinc-finger protein (PLZF) is a transcriptional repressor. To investigate the role of PLZF in the regulation of cytoadhesion molecules involved in the mobilization of hemopoietic cells, we have analysed PLZF and very late antigen 4 (VLA-4) expression in normal and leukemic cells. In hematopoiesis, we found a negative correlation between PLZF and VLA-4 expression, except for the megakaryocytic lineage. In contrast, we observed a positive correlation between PLZF and VLA-4 expression in a panel of acute myeloid leukemia (AML) samples. In K562 cells expressing PLZF (K562-PLZF), we found that the expression of VLA-4 and c-kit was downmodulated. We have investigated the possibility for VLA-4 or the c-kit receptor to be direct target genes of PLZF in K562-PLZF cells and identified a PLZF DNA-binding site within the VLA-4 promoter. Furthermore, decrease in VLA-4 expression was associated with loss of adhesion on fibronectin-coated plates, which promotes drug-induced apoptosis of K562-PLZF cells. Our findings indicate that VLA-4 is a potential target gene of PLZF. However, in primary AMLs the control of PLZF on VLA-4 expression is lost. Altogether, we suggest that VLA-4 modulation by PLZF may represent an important step in the control of normal and leukemic cell mobilization.

MicroRNA-146a and AMD3100, two ways to control CXCR4 expression in acute myeloid leukemias.

CXCR4 is a negative prognostic marker in acute myeloid leukemias (AMLs). Therefore, it is necessary to develop novel ways to inhibit CXCR4 expression in leukemia. AMD3100 is an inhibitor of CXCR4 currently used to mobilize cancer cells. CXCR4 is a target of microRNA (miR)-146a that may represent a new tool to inhibit CXCR4 expression. We then investigated CXCR4 regulation by miR-146a in primary AMLs and found an inverse correlation between miR-146a and CXCR4 protein expression levels in all AML subtypes. As the lowest miR-146a expression levels were observed in M5 AML, we analyzed the control of CXCR4 expression by miR-146a in normal and leukemic monocytic cells and showed that the regulatory miR-146a/CXCR4 pathway operates during monocytopoiesis, but is deregulated in AMLs. AMD3100 treatment and miR-146a overexpression were used to inhibit CXCR4 in leukemic cells. AMD3100 treatment induces the decrease of CXCR4 protein expression, associated with miR-146a increase, and increases sensitivity of leukemic blast cells to cytotoxic drugs, this effect being further enhanced by miR-146a overexpression. Altogether our data indicate that miR-146a and AMD3100, acting through different mechanism, downmodulate CXCR4 protein levels, impair leukemic cell proliferation and then may be used in combination with anti-leukemia drugs, for development of new therapeutic strategies.

PLZF-mediated control on c-kit expression in CD34(+) cells and early erythropoiesis.

The promyelocytic leukemia zinc-finger protein (PLZF) is a transcription factor and c-kit is a receptor tyrosine kinase associated with human disease, particularly in hematopoietic cells. MicroRNAs (miRs) are post-transcriptional regulators of gene expression, and c-kit has been described as a target of miRs-221 and -222 in erythropoiesis. In the present study, we identified c-kit as a target of PLZF in normal and leukemic cells. Particularly, in erythropoietic (E) culture of CD34(+) progenitors, PLZF is downregulated, whereas c-kit expression at both the mRNA and protein levels inversely increases during the first days of E differentiation. In functional experiments, PLZF transfection induces c-kit downregulation, inhibits E proliferation and delays differentiation, whereas PLZF knockdown induces opposite effects, independently of miRs-221 and -222 expression. The inverse correlation between PLZF and c-kit expression was found in normal CD34(+)38(+/-) hematopoietic progenitor/stem cells and in acute myeloid leukemias of M0/M1 French-American-British subtypes, suggesting that the control of PLZF on c-kit expression may be crucial at the level of the stem cell/progenitor compartment. Altogether, our data indicate a new mechanism of regulation of c-kit expression that involves a transcriptional control by PLZF in CD34(+) cells and early erythropoiesis.